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Figure 2: Methods of identifying substrate channelling. (a) A reaction scheme and depiction of transient time (t) analysis based on data from a channelled bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) and a freely diffusing nonfunctional TS and DHFR (data from [7]). (b) Comparison of residual activity of a channelled or freely diffusing enzyme pair in the presence of a competing enzyme, for example, the malate dehydrogenase and citrate synthase couple in the presence or absence of alanine aminotransferase which competes for the metabolic intermediate (data from [7]). (c) Comparison of residual activity of a channelled or freely diffusing enzyme pair in the presence of an inhibitor of the second enzyme, for example, the inhibition of the TS-DHFR cascade by the inhibition of DHFR by pyruvate (data from [7]). (d) Enzyme buffering analysis of channelling; this approach is typically applied for following the channelling of NADH which assesses if the second enzyme of a couple can use bound as well as free NADH and is based on comparison of the reaction velocities following dramatic decreases in the size of the free NADH pools as represented in the scheme. If the enzyme is not able to utilize bound NADH the system is essentially just buffering NADH added to it hence the name (data from [7]). (e) Schematic representation of the isotope dilution experiment to assess the channelling of glycolysis. 13C labelled glucose was fed to isolated potato mitochondria and the label accumulation in succinate was monitored. Nonlabelled G6P, F6P, F1,6BP, DHAP, or GAP was separately added to the medium following the fractional enrichment in succinate reaching steady state [18]. (f) The result of isotope dilution experiments for F1,6BP. The time course plots showing the fractional 13C enrichment in DHAP following the addition of F1,6BP at 0 min. Data come from [18].